Takeuchi and Higashiyama (2012) identified the AtLURE1 defensin-like peptide cluster as diffusible, species-preferential pollen-tube attractants secreted by synergid cells of the Arabidopsis ovule, and posed five unresolved issues: the pollen-tube receptor(s) were unknown; additional redundant attractants were undefined; how central-cell CENTRAL CELL GUIDANCE (CCG) activity and MAA3-dependent RNA metabolism non-cell-autonomously regulate synergid attractant expression and secretion was unresolved; which downstream fertilization steps impose the strongest species specificity and their partner molecules were unknown; and the evolutionary forces shaping AtLURE1 gene turnover and polymorphism were untested. The first two issues have been substantially resolved in the subsequent literature (PRK6 and the MDIS1-MIK1/MIK2 receptor heteromer perceive AtLURE1; XIUQIU1-4 and ACC were identified as additional attractants, and knockout of the whole AtLURE1 family is fertile, reframing AtLURE1 as a conspecific pollen-tube emergence accelerator). The surviving open problem comprises three linked components: (1) the molecular mechanism, especially the mobile intercellular mediator and the RNA-level gate, by which the central cell and MAA3-dependent RNA metabolism control synergid AtLURE1/XIUQIU production and polar secretion; (2) a determination of which post-attraction steps (pollen-tube reception, induced rupture, sperm discharge) impose the strongest species-specific barriers, together with the partner molecules mediating them; and (3) identification of the evolutionary forces (selection, balancing selection, or neutral drift with demography) driving AtLURE1 cluster turnover and its accession-level polymorphism.

Background

In flowering plants, the immotile sperm cells are delivered to the female gametes by the pollen tube, a tip-growing cell that navigates from the stigma to an ovule and ruptures inside it to release the two sperm cells for double fertilization. The final navigation step, micropylar guidance, is controlled by the female gametophyte (embryo sac, the seven-celled haploid tissue containing the egg apparatus): two synergid cells flanking the egg cell secrete diffusible chemoattractant peptides that guide the pollen tube through the micropyle, the ovule's opening. The major attractants are cysteine-rich peptides (CRPs) of the defensin-like (DEFL) subgroup; the first such attractants, LUREs, were isolated from Torenia fournieri synergid cells (Okuda et al. 2009). In Arabidopsis thaliana, Takeuchi and Higashiyama (2012) showed that a species-specific tandem cluster of six DEFL genes, AtLURE1 (the sole species-specific DEFL cluster relative to the close relative Arabidopsis lyrata), is expressed specifically and abundantly in synergid cells, that AtLURE1 peptides diffuse onto the funiculus (the stalk attaching the ovule to the placenta) through the micropyle, that RNAi knockdown partially impairs micropylar guidance, and that recombinant peptides attract pollen tubes species-preferentially in vitro. Attractant expression depends on the synergid transcription factor MYB98 (Kasahara et al. 2005; Punwani et al. 2008). Strikingly, ovules mutant for CENTRAL CELL GUIDANCE (CCG), a TFIIB-like transcriptional regulator expressed in the central cell, the largest cell of the embryo sac and sister of the egg, whose fertilization gives rise to the endosperm (Chen et al. 2007), or for MAA3, a Sen1-like RNA helicase (Shimizu et al. 2008), fail to secrete AtLURE1 even though AtLURE1 is expressed in the synergid, implying non-cell-autonomous control of the synergid by the central cell and an RNA-metabolism-dependent gate on attractant secretion. The 2012 paper closed by posing five unresolved issues: receptor identity, additional attractants, CCG/MAA3 regulation, species specificity of the post-attraction steps, with FERONIA, a synergid-localized receptor-like kinase required for pollen-tube reception and rupture, and the maize defensin-like peptide ZmES4, which opens a pollen-tube potassium channel to trigger pollen-tube burst (Amien et al. 2010), named as candidate mediators, and the evolutionary forces behind AtLURE1 turnover. Later literature transformed two of these. The pollen-tube receptors for AtLURE1 were identified in 2016 as PRK6, a pollen-specific leucine-rich-repeat receptor kinase that asymmetrically accumulates at the tube tip toward an AtLURE1 source and signals through ROPGEF proteins, activators of ROP small GTPases that control tip growth (Takeuchi and Higashiyama 2016), and independently as the MDIS1-MIK1/MIK2 heteromer, a complex of pollen-tube receptor-like kinases whose extracellular domains bind AtLURE1 (Wang et al. 2016); the structural basis of AtPRK6-LURE1.2 recognition was solved in 2017 (Zhang et al. 2017). Zhong et al. (2019) then found that a septuple knockout of the entire AtLURE1 family is fully fertile: AtLURE1 is not required for conspecific fertilization but accelerates conspecific pollen-tube emergence from the transmitting tract, the ovarian tissue through which pollen tubes grow toward the ovules, acting as a prezygotic reproductive-isolation barrier, while the newly identified synergid peptides XIUQIU1-4 attract pollen tubes regardless of species; Liu et al. (2021) confirmed that AtLURE1/PRK6 signaling additionally promotes conspecific micropylar attraction, and Mou et al. (2020) identified the ethylene precursor ACC (1-aminocyclopropane-1-carboxylic acid) as a further ovular attractant signal that promotes LURE1.2 secretion. In contrast, three of the five issues remain without a settled answer. The regulatory question is only partially mapped: CCG operates with the CCG-binding protein CBP1, the Mediator transcriptional co-activator complex, RNA polymerase II, and central-cell AGAMOUS-like (AGL) MADS-box transcription factors to co-regulate LURE1 and other CRPs (Li et al. 2015); spliceosome subunits PRP8A/PRP8B are required for MYB98/LURE/CRP expression in ovules (Kulichová et al. 2020); F-actin polarizes AtLURE1.2 secretion at the filiform apparatus, the elaborate wall ingrowth at the synergid apex through which attractants are released (Susaki et al. 2023); and FERONIA-dependent nitric oxide modifies LURE1 after pollen-tube arrival to shut off attraction (Duan et al. 2020). But the mobile signal that carries the central-cell cue to the synergid, and the molecular output of MAA3-dependent RNA metabolism, are still unidentified. For the species-specificity question, N-glycosylation by the ARTUMES/OST3/6 subunit of the oligosaccharyltransferase complex, the endoplasmic-reticulum enzyme that attaches N-linked glycans to secreted proteins, specifically gates interspecific pollen-tube reception (Müller et al. 2016), and the mechanics of reception have been clarified (Zhong et al. 2022; Gao et al. 2022; Lan et al. 2023 uncovered an antagonistic-RALF intergeneric barrier at the stigma — RALF, Rapid Alkalinization Factor, being a family of small secreted peptides — an earlier stage than the micropylar steps); yet no study ranks the post-attraction steps by barrier strength or identifies the partner molecules for rupture and sperm discharge, and the receptors for the non-AtLURE1 attractants, including XIUQIU, remain unknown (Nagae et al. 2022). Finally, no dedicated population-genomic study of the AtLURE1 cluster has appeared: the 2012 finding of rapid turnover with no detected positive selection (interpreted as neutral drift, with cysteine substitutions and stop codons segregating among accessions) still lacks a test with broad population sampling and functional assays of allele pairs. A recent review (Takeuchi 2021) summarizes the action, regulation, receptors, and evolution of LURE-type CRPs and confirms that these questions drive current work in the field.

Problem Statement

Determine the remaining molecular and evolutionary account of synergid-derived pollen-tube attraction in Arabidopsis thaliana, as posed by Takeuchi and Higashiyama (2012) and surviving the 2016-2023 literature. Three linked components jointly serve this objective. (1) Non-cell-autonomous regulation of attractant production and secretion: identify the molecular mechanism by which CCG/CBP1 transcriptional activity in the central cell (the largest cell of the embryo sac) controls AtLURE1 and XIUQIU expression and polar secretion in the synergid cells — in particular, the intercellular mediator that carries the central-cell cue to the synergid — and identify the RNA-level mechanism, downstream of or parallel to the MAA3 Sen1-like RNA helicase, that gates synergid attractant secretion despite intact expression. (2) Species specificity of the post-attraction steps: establish, by comparative assessment of pollen-tube recognition/reception, induced pollen-tube rupture, and sperm discharge, which of these steps impose the strongest species-specific barriers to interspecific fertilization, and identify the partner molecules that mediate those barriers. (3) Evolutionary dynamics of the AtLURE1 cluster: determine which evolutionary forces — directional or balancing selection, neutral drift, gene conversion, or demographic processes — shaped the species-specific formation and maintenance of the AtLURE1 cluster and its polymorphism among A. thaliana accessions (including cysteine substitutions and stop mutations). An answer may resolve the components separately, but each must be evaluated against the 2012 source question without narrowing it (for example, an answer to (2) must compare the post-attraction steps, not only the already-characterized micropylar attraction step).

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